The over- and under-expression of certain proteins in extracellular vesicles has been observed in many physiological and pathological conditions; however, a simple method to sort vesicles based on contrast in protein content is yet to be developed. We herein present a nonaffinity-based method for rapid and inexpensive isolation of lipid vesicles based on their membrane protein content. Based on a composition-specific thermophysical property change of vesicles at different protein contents, an acoustic property change that enabled an acoustophoretic separation was observed. This change was demonstrated in a thermally modulated acoustofluidic device in the form of a shift in vesicle migration from the nodal plane to antinodal plane at a specific temperature known as the acoustic contrast temperature (TΦ). Using phosphatidylcholine vesicles containing the membrane proteins gramicidin D, alamethicin, and melittin at molar contents ranging from 0.001% to 10%, we observed that increasing the membrane protein content brought about conformational changes in the membrane which afforded the vesicles distinctive acoustic properties. Then, by establishing an acoustic contrast temperature window, vesicles with the same protein but different molar content were successfully separated. The efficiency of the separation was studied for various vesicle mixtures and a separation efficiency as high as 97% was accomplished. In order to confirm the technique's applicability for biological samples, sheep red blood cells with various melittin peptide contents similarly demonstrated the depressing effects of melittin on membrane bending modulus and depressed the TΦ of the cells. This method holds promise for a myriad of applications in the biomedical field, especially in bioanalytical research.